Project Summary Quercetin, a plant flavonoid is a potent antioxidant and anti-inflammatory agent. We have performed preclinical studies assessing the suitability of quercetin in the treatment of chronic obstructive pulmonary disease (COPD), a third leading cause of death in the U.S. We showed that quercetin reduces lung inflammation, goblet cell metaplasia and progression of emphysema, in part by increasing expression of the deacetylase Sirt1 in elastase/LPS-exposed mice displaying typical features of COPD. Our pilot studies indicate that elastase/LPS-exposed mice show reduced nuclear FOXO3a, a transcription factor that negatively regulates inflammation and provide resistance to oxidative stress. Further, we found that COPD airway epithelial cells which maintain the phenotypic characteristics of the COPD airway epithelium also show reduced nuclear levels of Sirt1 as well as FOXO3a. Infection with human rhinovirus (RV), a common cause of COPD exacerbations further reduced nuclear Sirt1 and FOXO3a levels. Our pilot studies also show that quercetin promotes translocation of FOXO3a to the nucleus in elastase/LPS-treated mice. Further, quercetin treatment of COPD epithelial cell cultures reduced IL-8 expression and mucus metaplasia, while restoring nuclear FOXO3a levels. Finally, quercetin improved viral clearance after RV challenge in both elastase/LPS mice and COPD epithelial cells. The overall goal is to elucidate the mechanisms by which quercetin modulates nuclear FOXO3a and Sirt1 levels, thereby decreasing inflammation, goblet cell metaplasia and augment viral clearance in COPD models. To accomplish this goal, we propose the following specific Aims. 1. Determine the mechanisms by which quercetin modulates FOXO3a activity and inhibits overexpression of IL-8 and other chemokines and reduces lung inflammation in COPD models. Nuclear translocation of FOXO3a is regulated by phosphorylation and acetylation. We test the hypotheses that 1) quercetin treatment blocks Akt kinase activity, thereby increasing FOXO3A translocation to the nucleus; 2) quercetin-induced Sirt1 deacetylates nuclear FOXO3a, preventing phosphorylation by Akt export from the nucleus; and 3) Sirt1 and FOXO3a together block NF-?B binding to the CXCL-1, 2, 5 and 8 promoter, thereby attenuating cytokine expression and lung inflammation. 2. Examine the mechanisms by which quercetin decreases goblet cell metaplasia in COPD airways. Our pilot data indicate aberrant activation of EGFR in COPD cells as well as in COPD mouse model. We will test the hypotheses that 1) quercetin-induced nuclear FOXO3a positively regulates expression of E-cadherin, which sequesters EGFR at the epithelial cell basolateral surface; 2) quercetin directly inhibits EGFR activity; 3) quercetin, by restoring Sirt1 levels, inhibits matrix metalloproteinase (MMP) expression, thereby decreasing the availability of EGF ligands; and 4) reduced EGFR activity decreases expression of mucin genes, preventing goblet cell metaplasia. 3. Determine the mechanisms by which quercetin increases viral clearance in COPD airways. COPD exacerbations are associated with increased oxidative stress We will test the hypotheses that: 1) oxidative stress induces formation and accumulation of autophagosomes, providing a platform for viral replication; 2) RV infection, via its effects on Akt and CBP/p300, decreases nuclear FOXO3a and phase II antioxidant enzyme expression, amplifying derangements present in COPD cells; and 3) quercetin restores antioxidant enzyme expression by modulation of FOXO3a and Sirt1, decreasing autophagy and increasing viral clearance. Completion of these studies will provide important insights into mechanisms by which quercetin decreases airway inflammation, goblet cell metaplasia and viral clearance in chronic obstructive lung disease. These studies are required to determine the suitability of quercetin as an alternative complimentary medicine in the management of COPD lung disease.